The existence of naturally-occurring macromolecular species known as "antifreeze proteins" or "thermal hysteresis proteins" and the subclassifications "antifreeze glycoproteins" and "antifreeze polypeptides" is well known and widely reported in the literature. The discovery of antifreeze glycoproteins, for example, was first reported by DeVries, A. L., et al., in "Freezing Resistance in Some Antarctic Fishes," Science 163:1073-1075 (Mar. 7, 1969). DeVries, et al. observed that water temperatures in McMurdo Sound, Antarctica, average -1.87.degree. C. over the year, and that various species of fish survive in these conditions despite the fact that the total concentration of sodium chloride and other low molecular weight substances present in the blood sera of these fish could only produce a freezing point depression of less than half that needed for survival under these conditions. While earlier studies had indicated that the survival of these fish might be attributable to the presence of certain macromolecular antifreeze compounds in the blood of the fish, DeVries and his coworkers were the first to establish the nature and composition of these macromolecular species. With typical molecular weights ranging from about 2,500 to about 34,000, these species are now referred to as antifreeze glycopeptides or "AFGPs." Further investigations have revealed that many species of north temperate and Arctic fishes carry antifreeze compounds in their blood. Some of these compounds are glycoproteins while others contain no sugar moieties and are referred to as antifreeze polypeptides or "AFPs," having molecular weights ranging from about 3,300 to about 12,000.
Some of the common features of AFGPs and AFPs are as follows:
(a) They are present in relatively large concentrations in fish blood (about 10-40 mg/mL). In fish that experience seasonal changes in temperature, the compounds are present in low concentrations, or are absent entirely, throughout much of the year, increasing to higher levels during seasonal periods of low water temperature and low photoperiod. In fish residing in Antarctic regions where the waters remain at subfreezing temperatures, the compounds remain present throughout the year, in high concentrations. PA1 (b) The compounds achieve freezing-point depression in a non-colligative manner (i.e., the effect varying with molecular size but not the number of molecules present) and to a far greater degree than one would expect on the basis of the osmolality of the solution containing the molecules. PA1 (c) While the compounds lower the freezing point, the melting point remains unaffected (i.e., they exhibit thermal hysteresis behavior). PA1 (d) The compounds function by interacting with and modifying the morphology of ice crystals. PA1 (a) the isolation and characterization of the proteins, PA1 (b) the conformations of the protein molecules, including second and higher order conformations, PA1 (c) the interaction of the protein molecules with ice, and PA1 (d) means of preparing the proteins synthetically including methods involving the use of recombinant DNA. PA1 (i) hypothermal conditions defined by temperatures above the normal freezing point of water (0.degree. C.), and therefore with no possibility of ice formation, and below the physiological temperature of the cells; PA1 (ii) vitrification conditions defined by temperatures at or below the glass formation (or glass transition) temperature, such as for example from 150K down to about 4K, and by the presence of vitrifying agents which promote vitrification and inhibit crystallization; PA1 (iii) freezing conditions, such as temperatures from the normal freezing point of water down to about 4K, which permit the formation of ice crystals; PA1 (iv) hyperthermal conditions defined by temperatures above the physiological temperature of the cells, for example temperatures within the range of the physiological temperature up to about 10.degree. C. above the physiological temperature; and PA1 (v) conditions defined by chemical environments which differ from the physiological chemical environment of the cells, such as conditions of nonphysiological pH and other variations from the physiological chemical composition, as well as such conditions in combination with conditions of nonphysiological temperature.
Because of the thermal hysteresis effect, the compounds are generically referred to herein for convenience as "thermal hysteresis proteins."
Prior to the present invention, studies relating to thermal hysteresis proteins have focused on
The only investigations into biological functions and utilities of the proteins are the studies of the interaction of the protein molecules with ice. These studies have addressed such matters as the thermodynamics and surface kinetics of the ice crystal surfaces, the direction of crystal growth, and the modes and directions by which the proteins block the crystal growth.
Shortly after the discovery of the proteins, attempts were made to exploit the proteins' antifreeze character by using them in biological materials other than those of the fish from which they were derived. As an example, red blood cells were treated with the proteins using standard cryopreservation procedures and exposed to freezing conditions. The results were not favorable, however, since the proteins caused the complete destruction of the cells rather than their preservation. There has been no evidence that the proteins could be successfully used with biological materials other than the fish to which they were native.
In summary, prior to the present invention no studies have been performed on properties or utilities of the proteins in contexts unrelated to ice crystals. Nor have there been studies which have demonstrated or suggested any potential benefit that these proteins might offer to organisms other than fish, and particularly to mammalian organisms and tissues. Furthermore, no studies have been conducted which suggest that these proteins might have any beneficial effect at all at concentrations significantly below the concentrations in which they are found in the fish. In fact, the well-recognized and extensively reported non-colligative character of these proteins discourages any suggestion that either injurious or beneficial effects attributable to these proteins might vary with concentration.